Typical spectrophotometric sample analysis systems require that multiple, single wavelength electromagnetic waves be applied to a sample solution sequentially during an analyte detection and quantification procedure to, for instance, detect and quantify multiple analytes or an analyte and contaminants present therein. That is multiple, single wavelength electromagnetic waves in a beam of electromagnetic waves can not be simultaneously caused to pass through a sample solution and the energy absorbed by said sample solution from each single wavelength electromagnetic wave simultaneously detected, analyzed, displayed and/or recorded. A user of typical spectralphotometric systems must then typically change monochrometers and filters in a typical spectrophotometric system between analysis steps to provide sequentially, the electromagnetic wave single wavelengths required. Use of such systems is thus tedious and time consuming and the results provided thereby are prone to error. It is emphasised that a system which would allow immediate simultaneous analysis, display and/or recording of energy absorbed from a multiplicity of single wavelength electromagnetic waves which are caused to simultaneously pass through a sample as a beam of electromagnetic waves, would greatly increase convenience of use and improve accuracy of achieved analysis results.
In addition, most sample analysis systems have limited capability regarding detecting and quantifying analytes dissolved in small volume sample solutions, (on the order of a few microliters or less), and small sample solution volumes must often be diluted to provide sample solution volumes sufficiently large to fill sample retaining volumes present therein. Of course, dilution reduces the concentration of the analytes in a sample solution, and said reduction of analyte concentration makes it more difficult for the sample analysis system to detect and quantify said analytes.
Continuing, while the present invention is not limited to use with nucleic acids, (eg. DNA, RNA), and proteins, said sample analytes are at times particularly difficult to obtain in solution volumes greater than a few microliters. The following discussion uses nucleic acid and proteins as an example to further describe the above identified problem.
There are two basic approaches to detecting and quantifying nucleic acids in a sample:
1. Ethidium Bromide Fluorescence, applicable to DNA, and PA1 2. Spectrophotometry, applicable to DNA & RNA. PA1 1. 1.8 for DNA, and PA1 2. 2.0 for RNA.
It is mentioned in passing that if the sample analyte is DNA, an approach, as indicated, to quantifying the amount present is by use of a technique which uses Ethidium Bromide. Said technique is applicable to small volumes of sample. Said technique provides that double strand DNA be subjected to treatment with Ethidium Bromide, which Ethidium Bromide interacts with the double stranded DNA to become intercalated therein. When Ultraviolet light is then caused to illuminate the DNA-Ethidium Bromide combination, fluorescence occurs. The amount of fluorescence is directly proportional to the amount of DNA present. As alluded to, however, this technique is not applicable to single stranded RNA.
The spectrophotometric sample analyte detection and quantification technique as applied to nucleic acids requires measurement of the amount of energy absorbtion from two single wavelength, (eg. two-hundred-sixty (260) and two-hundred-eighty (280) nanometers), electromagnetic waves which are caused to pass through a volume of sample solution which contains nucleic acid analytes. Energy absorbtion at two-hundred-sixty (260) nanometers is proportional to the quantity of nucleic acids present and that at two-hundred-eighty (280) nanometers is an indication of the presence of protein or phenol contaminants, for instance. In the absence of protein or phenol the ratio: EQU (Absorbtion at 260 nm)/(Absorbtion at 280 nm)
is typically not significantly lower than:
If a ratio value lower than indicated is obtained, energy absorbtion interference at two-hundred-sixty (260) nanometers is indicated and the nucleic acid purity of the sample under analysis is questionable.
The above discussion provides insight to the fact that, particularly in the area of where nucleic acids and proteins are the analytes present in a sample solution, a need exists for a sample analysis system, preferable a spectrophotometric sample analysis system, which can provide sample analyte quantification analysis, especially when only a few microliters or less of relatively concentrated analyte containing sample solution are available. It is noted, however, that sample analytes other than nucleic acids and proteins are often available in only very small solution volumes as well, and that spectrophotometric analysis techniques of determining the quantity of sample analyte present therein can be equally applicable thereto. Typically, wavelengths other than two-hundred-sixty (260) and two-hundred-eighty (280) nanometers are used when sample anlaytes other than nucleic acids are present, but the spectrophotometric sample analysis technique is unchanged. That is, the simultaneous determination of the energy absorbtion from each of two or more single wavelength electromagnetic waves, in a beam of electromagnetic waves, as it is passed through a sample analyte containing solution, is identifying of the quantity of certain sample analytes present therein.
A Search for Patents which describe spectrophotometric sample solution analysis systems for use in analysis of analytes in microliter volumes of sample solutions provided no Patents directly focused on analysis of small volumes of nucleic acid or protein solutions. Patents which describe the use of sample flow cell systems, a Patent which describes a small volume (eg. 1 microliter), pumping system and Patents which describe measurement of absorbtion of one or more signals issuing from waveguides in which are present analytes, however, were identified.
A Patent to Johansson, U.S. Pat. No. 4,294,802 describes a system which is capable of feeding a number of small volume (eg. one (1) microliter), sample solution boluses, simultaneously through a number of parallel flexible hoses.
A Patent to Riley, U.S. Pat. No. 4,610,544 describes a system for entering reagent and sample into a flow channel of preferably not greater than one (1) milimeter internal diameter. A single peristalic pump is used to draw reagent and sample into separate arms of the flow channel, which merge. A carrier liquid is sequentially drawn into the flow channel to carry along a discrete liquid slug of mixed reagent and sample. The flow cell is generally sufficiently long to allow reagent and sample to react with one another prior to entering an analytical station, such as a colorimiter or spectrophotometer. The Background Section in Riley describes prior discrete analyzers in which a sample is placed in an individual container during analysis. In addition, systems which mix, and retain for a designated time, sample and reagent in a chamber and then pass said mixture to a measurement sell such as a photometric measurement system are alluded to. Systems in which multiple samples are caused to flow sequentially along a flow channel with or without intervening separators such as slugs of air are also mentioned. Flow injection systems in which the carrier medium is the reagent and into which is injected precisely measured amounts of sample by syringe through a septum, or a rotary valve are also mentioned.
A Patent to Kuroishi, U.S. Pat. No. 4,582,687 describes a system for performing rate assay analysis. Sample and reagent are introduced into a stream of carrier liquid and transferred through a series of flow cells. Said series of flow cells are positioned such that a single beam of light simultaneously passes through each. The reagent and sample enters a first flow cell early in the reaction process between said reagent and sample and the absorbtion of the light beam thereby is monitored. A delay line, (eg. a long coil reaction tube), carries the reacting reagent and sample into the next flow cell, after all such reagent and sample has left the first flow cell, and again the absorbtion of the light beam is monitored. The difference in absorbtion factors is related to the rate of the reaction between the reagent and the sample. The period of time between the reagent and sample entering the first and a subsequent flow cell is determined by the length of the delay line.
A Patent to Yamamoto, U.S. Pat. No. 4,398,894 describes a system for quantitatively determining the degree of agglutination of particles. A liquid which contains agglutinated clots is made to slowly transfer through a small tube. In the course of said transfer agglutinated clots and nonagglutinated particles separate from one another in the liquid. The difference between optical properties of the leading edge, (ie. agglutinated layer), and trailing edge, (ie. non-agglutinated particles), of a transferring agglutinated clot and surrounding liquid provides a quantification of the degree of agglutination present. Optical properties are detected by photometric means at multiple locations along the small tube.
A Patent to Sutherland et al., U.S. Pat. No. 4,775,637 describes an immunoassay apparatus having two waveguides. One waveguide has its internal surface coated with a reactant specific to a component for which analysis is desired. The other waveguide does not have such a coating present. Dual beams are generated from a monochromator and one of said beams is caused to pass through one of said dual waveguides, and the other of said beams is caused to pass through the other of said dual waveguides. Measurement of relative absorbtion of the two beams provides information regarding the presence of a component which reacts with the reactant present on the internal surface of one of the waveguides. Simultaneous monitoring of two signals is thus an important aspect of the operation of the invention. However, use of two different wavelengths is not taught. A second Patent to Sutherland et al., U.S. Pat. No. 4,818,710 does suggest the use of two or more wavelengths, but requires that one or more coatings of reactants specific to species to be analyzed be present. A light signal carried by a waveguide undergoes interaction with a bulk analyte and leads to development of a first signal. Simultaneously said light signal interacts with a layer of complex which results from the reaction of one or more specific reactants with one or more species to be analyzed, leading to development of a second, (or more) signal. When a second wavelength is used in the light signal, the waveguide is coated with different specific reactants at different locations along it length. The two, or more, different wavelength signals developed are separated for analysis by band-pass, dichromatic beam splitters etc. Sample entry through a flow cell is taught. Signals to be analyzed can be developed by absorbtion, scattering or the generation of fluorescence.
A Patent to Buckles, U.S. Pat. No. 4,399,099 describes a system which employs an energy transmissive core and employes one or more sheaths which selectively absorb, react with, and/or filter an analyte or product of an analyte. The passage of energy through the energy transmissive core is modified by reason of events which occur in one or more of the sheaths. If no sheath is present, events which occur in an ambient fluid serve as energy passage modifiers. The resulting modification of transmitted energy is monitored and serves as a measure of analyte. The energy may be present in electromagnetic form, electrical or sonic form. The Buckles teachings are that two or more such systems can be used simultaneously to simultaneously measure more than one analyte.
A Patent to Shanks et al., U.S. Pat. No. 4,810,658 describes a method for analyzing small liquid samples optically to discriminate sample material which is bound to a solid surface from sample material which remains free in solution. Light is entered into a transparent solid optical waveguide surface, which surface is perpendicular to the optical axis of said waveguide and to which surface is bound sample material. Light emerging from the end of the waveguide within a certain angle with respect to the optical axis of said waveguide is measured. The emerging light provides the desired information. While the limiting sample solution volume which can be analyzed by the system is not specifically designated, mention of a Patent G.B. No. 2,090,659 in the Background Section of the Patent indicates that a sample size of more than ten (10) microliters is required.
From the above it can be concluded that while prior Patents teach systems and methods which use flow cells, pump small volumes, (eg. one (1) microliter), of sample solution, simultaneously utilize multiple light beams and light beams comprised of more than one wavelength to provide sample analysis; a need still exists for simple to use Spectrophotometric sample analysis system which can "simultaneously" quantify the amount of nucleic acids, proteins and/or other sample analytes which are present in a sample solution, when the sample solution volume available for analysis is on the order of a few microliters or less. Said system should have the capability to analyze a small volume of sample solution for the presence and quantity of many sample analytes essentially simultaneously, and instantaneously display and/or record said analysis results.